Project 4's objective is to characterize transcriptional and epigenetic mechanisms underlying heroin abuse in humans, with complementary, mechanistic studies in rodents. We focus on two key brain reward regions, nucleus accumbens (NAc) and prefrontal cortex (PFC), and concentrate on dysregulation of genes related to glutamatergic neurotransmission, a major focus of Projects 1 and 2. By studying human brain, the proposed studies fill a major gap in the field. Preliminary data from a microarray analysis have already demonstrated abnormal expression of several glutamate-related genes in NAc of human heroin abusers. We will validate and extend these findings by obtaining a more comprehensive view of gene expression in NAc by use of RNA-Seq in conjunction with the Chromatin and Gene Analysis Core, and will study the PFC as well. Specific glutamate-related genes will be further analyzed with respect to the neuronal cell types that show abnormal expression within these regions of human brain using laser capture microdissection. To interrogate the molecular mechanisms underlying these heroin-induced molecular changes, we will perform parallel studies in rats that self-administer heroin over an extended time period, and utilize viral gene transfer to evaluate causal relationships between specific gene targets and heroin seeking and taking. These studies of gene expression will then be extended further to the epigenetic level to gain unique insight into the underlying mechanisms involved. ChlP-Seq will be conducted, again with the Core, to map genome-wide binding of a select number of chromatin modifications and transcription factors in the human NAc and PFC. For example, we will initially explore H3K9me2 (dimethylation of Lys9 of histone H3) and the transcription factor CREB, given the wealth of evidence from Projects 1 and 3 for their involvement in drug addiction. We will use advanced bioinformatics to align our data from human and rodent models and characterize biochemical pathways that display dysregulation. Overall, the novel research in this Project, which is fully integrated with the other Projects and Cores, will provide a crucial translational dimension to our overall program of research, and help advance molecular knowledge about the pathophysiology of drug addiction.